The present invention relates to apparatus for determining a property of a sample such as an enzyme sample. For example, the present invention may be used to determine the enzyme activity of the sample.
In connection with methods and apparatus for determining properties of the above type, various problems are encountered at the present time. In order to illustrate the problem solved by the present invention, the following example of enzyme activity value measurement may be considered.
The following reaction formula represents, for example, the reaction of activity value measurement of LDH (lactate dehydrogenase) which in a suitable serum forms a sample which is to be tested. LDH acts as a catalyzer for the reaction in the direction of the arrow: ##STR1##
While the actual reaction set forth above is reversible, in the case where use is made of a reagent containing pyruvic acid as a substrate and NADH as a coenzyme, the reaction equilibrium is directed in a pronounced manner toward the right, and during the initial time period when the reaction takes place, NADH is consumed in a rate-determining state so that the concentration of NADH diminishes at a constant rate. It is thus possible to measure the concentration of this NADH by measuring the extent to which ultraviolet light of a wavelength of, for example, 340 nm (nanometer) is absorbed when directed through the mixture of the sample and reagent while the above reaction is going forward. Thus the enzyme activity value can be represented by the rate of consumption or in other words the rate at which the concentration of NADH diminishes, which is to say the rate variation of absorbance of the light of the above wavelength with respect to time.
Thus, it is important that the real value of enzyme activity correspond to the variation of the light absorbance as set forth above.
Thus, with the present invention it is possible to provide for measurement of an enzyme activity value by way of a photoelectric colorimeter or a spectrophotometer, and in particular the present invention relates to reaction containers to bring about accurate control of the enzyme reaction temperature.
The possibility of measuring enzyme activity by utilizing ultraviolet light absorption by the coenzyme NADH has greatly advanced clinical biochemical examination and has greatly improved the technique of medical examination and treatment.
This enzyme activity value measuring method is characterized in that the reaction rate between a chemical substance forming a substrate and an enzyme which has a specific catalyzing action on the substrate can be directly and optically observed. In this way it has become possible to measure enzyme activity values according to a uniform method and in a rapid manner with respect to very many types of enzymes.
In practice, however, this kinetic assay method has various problems so that it cannot be readily adopted by many clinical examiners.
One of the problems is with respect to the time restriction of the rate-determining step measured only in the rate-determining state during the initial period of the reaction. The real value cannot be obtained if, as the reaction advances to the right with lapse of time, the reaction in the reversed direction commences or a reaction-alienated substance is produced. Moreover, the enzyme activity is influenced to a very high degree by temperature. For example, in the vicinity of 30.degree. C, it has a temperature gradient of 7% per 1.degree. C, so that for accurate measurement an extremely elaborate temperature control is required. Thus, in connection with enzyme activity value measurement, all of the elements constituting the reaction system must be controlled so as to have the predetermined temperature up to the time of initiation of the reaction, and after initiation of the reaction the temperature of the reaction system must be kept at a predetermined value until the measurement is completed.
In practice, however, this latter type of procedure is extremely difficult to achieve. For example, if the serum sample, the substrate and the NADH solution are separately heated until they reach the predetermined temperature, the temperature of the reaction system may be remarkably different from the predetermined value as a result of loss of heat caused through measurement by pipette, mixing, transfer to the absorption cell, etc.
In order to avoid difficulties of the above type, it has been proposed to compensate the measurement value with respect to the temperature at the time of reaction. Clinically, however, such procedures never result in reliable values due to the nature of the enzyme which is generally composed of several isomers.